In the fields of food sanitation, biology, clinical test, medical science, ultrapure water and environmental studies, it is common to analyze an intracellular component extracted from cells contained in a sample.
There are various purposes of analyzing the intracellular component, for example, for determining presence/absence, content and activity of the intracellular component, for amplifying nucleic acids, for determining microorganisms in a sample (e.g., determining presence/absence or types of microorganisms, and counting the number of live microbes).
A method for extracting the intracellular component is known in which a reagent for extracting the intracelluar component (hereinafter, referred to as an "extraction reagent") is added to a sample containing cells. Examples of the extraction reagent include ones containing, as an effective component, a detergent, trichloroacetic acid, lytic enzyme such as lysozyme, or ethanol.
Among the above-mentioned extraction reagents, the reagent containing a detergent is frequently used. According to a method that utilizes the extraction reagent containing a detergent as an effective component (hereinafter, referred to as a "detergent method"), examples of the detergent used include anionic detergents (e.g., sodium dodecyl sulfate (SDS), potassium lauryl sulfate, sodium monolauroyl phosphate, sodium alkylbenzenesulfonate), cationic detergents (e.g., benzalkonium chloride (BAC), benzethonium chloride (BZC), cetylpyridinium chloride, cetyltrimethylammonium bromide, myristil dimethylbezyl ammonium chloride), ampholytic detergents (e.g., Twittergent Detergent 3-08, 3-10, 3-12, 3-14, 3-16, Tego) and nonionic detergents (e.g., Tween 20, 60, 80, Span 60, 80, Triton X-45, X-100, polyoxyethylene ether, polyoxyethylene lauryl ether).
According to the detergent method, higher detergent concentration in the extraction reagent results in higher extraction efficiency of the intracellular component. However, higher detergent concentration causes poorer analysis sensitivity and accuracy, as the detergent will inhibit the step of analyzing the intracellular component.
A method for measuring microorganisms is known in which an intracellular component, adenosin triphosphate (ATP), is measured by a bioluminescence method. ATP measurement by the luminescence method is very useful in terms of easiness, short measurement time and high sensitivity. As the bioluminescence method, luciferin-luciferase luminescent reaction method is generally used.
When the detergent is used for extracting ATP in the method for determining the number of cells through luciferin-luciferase luminescent reaction, higher detergent concentration will increase the efficiency of extracting ATP but will also decrease analysis sensitivity and accuracy, as the luminescent reaction is inhibited. This is assumed to be caused by deactivation of enzyme luciferase upon contact with the detergent, which results in rapid decay of luminescence. On the other hand, lower detergent concentration can reduce the inhibition of luminescent reaction but will result in insufficient ATP extraction efficiency.
Intracelluar ATP can be extracted by adding an extraction reagent to a sample containing cells. Usually, the extraction reagent is added such that the final concentration of the detergent is around 0.005%. In order to bring out satisfactory extraction ability, higher final detergent concentration is preferable. However, when the final concentration of the detergent is high (for example, when the final concentration is 0.01% or higher), measurement sensitivity and accuracy are greatly deteriorated as the detergent significantly inhibits the luminescent reaction.
Furthermore, when a nucleic acid is to be amplified by a PCR method, a detergent will inhibit the PCR reaction and no PCR product would appropriately be formed.
When the intracelluar component is an enzyme, there is a problem that the extracted enzyme will be denatured or deactivated by the detergent.
As a substance to repress the interruption of the analysis step (e.g., bioluminescent reaction) caused by the detergent, a method utilizing cyclodextrin or a derivative thereof is known (Japanese National Phase PCT Laid-open Publication No. 6-504200). Another known method for measuring intracellular ATP comprises the steps of: bringing a sample containing cells in contact with a detergent to extract intracellular ATP; and measuring the ATP by luciferin-luciferase luminescent reaction technique, wherein the luminescent reaction technique is applied after bringing the ATP-extracted sample in contact with cyclodextrin (Japanese Laid-Open Publication No. 7-203995).
However, methods utilizing cyclodextrin have the following problems.
(1) Cyclodextrin itself inhibits luminescence: For example, when .alpha.-cyclodextrin exists in a luminescent reaction solution at a concentration of 1% or 2%, strong inhibition of 25% or 50% is caused, respectively.
(2) Cyclodextrin is expensive: .alpha.-cyclodextrin which has the most superior neutralizing ability of cyclodextrin costs about 20,000 yen per kilogram.
As described above, for a method utilizing an extraction reagent that contains a detergent as an effective component, it is a pressing need to establish a method to efficiently extract an intracellular component without inhibiting the step of analyzing the intracellular component (hereinafter, simply referred to as an "analysis step").
The term "analysis step" as used herein refers literally to a step of analyzing an intracellular component, to a pretreatment for the analysis, and further to any process performed after the step of extracting the intracellular component.